4. Discussion
The current study revealed, for the first time, that 10 μg/mL ox-LDL causes necroptosis in foam cells derived from VSMCs and that CTRP9 exerts a potent protective effect against ox-LDL-induced necroptosis. CTRP9 inhibited the formation of cell necrosomes and the subsequent inflammation in VSMCs pretreated with ox-LDL. Mechanistically, this effect is attributable to the upregulation of antioxidant enzymes due to AMPK activation in VSMC-derived foam cells. Importantly, CTRP9 overexpression significantly suppressed RIP3 and promoted the features of atherosclerotic plaque stability. These results imply that CTRP9 plays a protective role against atherosclerosis by regulating VSMC-derived foam cell necroptosis.
Ox-LDL acts as a danger-associated molecular pattern (DAMP) to stimulate the inflammatory activation of macrophages, VSMCs, and other surrounding cells [15]. In our study, ox-LDL potently induced cell necrosis and necroptosis in a dose-dependent manner at concentrations as low as 10 μg/mL. During the formation of atherosclerotic plaques, cellular turnover in the plaques becomes imbalanced as cells engraft lipids to form foam cells and undergo both apoptotic and nonapoptotic cell death [16]. Cell necroptosis leads to inefficient cell clearance, thus contributing to the acellular necrotic core. Cell necroptosis is characterized by the disruption of cellular membranes, release of massive intracellular contents, and expansion of the secondary inflammatory response, which may play a central role in plaque expansion. Therefore, considering that ox-LDL is abundantly present in plaques, our study provides a new perspective on the mechanism by which ox-LDL promotes plaque progression in atherosclerosis.
Necroptosis is closely associated with inflammation. We found that, consistent with the cellular characteristics of necroptosis, ox-LDL induced the secretion of proinflammatory cytokines such as IL-1β, IL-6, IL-18, and MCP-1. In addition, ox-LDL accelerated the expression of the macrophage marker CD36 and cell adhesion molecules. Interestingly, ox-LDL also induced the expression of CD47. CD47, known as the “do-not-eat-me” molecule, affects effective programmed cell removal and may lead to clonal expansion. Leeper et al. showed that VSMCs can produce hyperproliferative cells, which promote inflammation and escape immune surveillance, further leading to plaque expansion [6]. The pro-atherosclerosis factor TNF-α participates in the expression of CD47, which is upregulated in plaques [17]. In contrast, knockdown of the key antiphagocytic molecule CD47 suppressed the clonal expansion of SMCs in plaques in vivo [6]. CD47 expression in VSMC-derived foam cells after ox-LDL treatment may provide a clue to the potential mechanism underlying VSMC-derived foam cell accumulation in plaques and may represent a translational target for atherosclerosis treatment.
Oxidative stress response may be associated with ox-LDL-induced cell death [18,19], and a previous study has indicated that ROS overexpression induces necroptosis [20]. However, whether mtROS affect ox-LDL-induced necroptosis in VSMC-derived foam cells has not been reported. In this study, increased cell necroptosis accompanied by altered mtROS levels and ΔΨm was observed in ox-LDL-treated VSMCs, but adding a ROS scavenger effectively reduced cell necroptosis. The results confirmed that oxidative stress was the basic mechanism underlying ox-LDL-induced cell necroptosis. We also revealed that CTRP9 exerted a strong antioxidant effect and that CTRP9 pretreatment prevented the abnormal accumulation of mtROS and inhibited cell necroptosis induced by ox-LDL. Consistent with our results, Zuo et al. also reported that CTRP9 restored the ΔΨm and suppressed ROS generation [21]. Nevertheless, inhibition of the AMPK pathway in VSMC-derived foam cells by pretreatment with Compound C abolished the CTRP9-induced expression of antioxidant enzymes and downregulation of cell necroptosis-related proteins. These results indicate that AMPK activation is crucial for the protective effect of CTRP9 against ox-LDL-induced damage of VSMC-derived foam cells. Collectively, the findings of this study reveal the vital role of mtROS in ox-LDL-mediated necroptosis and the mechanism underlying the protective effect of CTRP9 against atherosclerosis.
RIP3 deficiency exerts athero-protective effects by reducing the lesion areas and plaque vulnerability [3,22]. Similarly, RIP1 inhibition via Nec-1 treatment reverses plaque maturation in Apoe−/− mice [4]. Knockdown of the necroptosis executioner MLKL decreases both programmed cell death and the necrotic core in plaques; however, the total lesion area remaines unchanged [16], suggesting that MLKL is associated with lipid accumulation in macrophages. In our study, we found that CTRP9 overexpression suppressed RIP3 expression in plaque and promoted the features of atherosclerotic plaque stability, suggesting that cell necroptosis is an effective target for atherosclerosis treatment. However, the therapies need to be carefully selected. For example, the advantage of RIP3 may be attributed to its function in regulating cytokine expression in VSMCs through a cell death-independent mechanism [23]; thus, a target that can inhibit both cell necroptosis and inflammation factor expression can improve clinical benefits. CTRP9 has multiple functions, including exhibition of anti-inflammatory activity, prevention of oxidative damage, and inhibition of cell necroptosis. Therefore, CTRP9 may be a potential drug target in atherosclerosis.